There is currently a surge of activity in developing virus-free lipid-based gene delivery systems for therapeutic applications because of their low toxicity, nonimmunogenicity, and ease of production. Cationic liposome-DNA (CL-DNA) complexes have shown gene expression in vivo in targeted organs, and human clinical protocols are ongoing. These lipid-gene complexes have the potential of transferring large pieces of DNA into cells. Indeed, partial fractions of order 1 million base-pairs of human artificial chromosome have been transferred into cells using cationic lipids as a vector although extremely inefficiently. Because our understanding of the mechanisms of action of CL-DNA complexes remains poor, transfection efficiencies are very low compared to gene delivery with viral vectors. The low transfection efficiencies with virus-free delivery methods are the result of poorly understood transfection-related mechanisms at the molecular and supramolecular levels, and a general lack of knowledge of interactions of lipid-gene complexes with components inside cells which lead to gene release and expression. The aims of this research application are (1) to clarify the relation between the physical and chemical parameters of CL-DNA complexes with a distinct nanostructure, and transfection efficiency in mammalian cells, and (2) to determine the nanostructures and transfection efficiency properties of a new class of surface-functionalized CL-DNA complexes, which are designed for specific interactions with cellular components. The structure of the lipid-gene complexes will be solved by using state-of the-art synchrotron x-ray diffraction techniques at the Stanford Synchrotron Radiation Laboratory. Laser scanning confocal microscopy will enable us to track the CL-DNA particles and observe their interactions with cells. The structures will be correlated to transfection efficiencies by modern molecular biology methods of quantitatively measuring expression of the luciferase reporter gene in mammalian cells. The broad long-range goal of the research is to develop optimal synthetic virus-free carriers of DNA for gene therapy and disease control.